Whole-exome sequencing of pathogenic genes in a family with congenital heart disease: A case report

Rationale: Congenital heart disease (CHD) is the most common birth defect and an important cause of noninfectious deaths in infants and children. It has high prevalence globally, placing an enormous burden on society and families. Studies of individuals with hereditary or sporadic CHD have provided strong evidence for its genetic basis. The aim of this study was to identify causative gene variants in a Chinese family with congenital heart disease. Patient concerns and diagnoses: Three generations of a CHD family were recruited. Proband III.9 was diagnosed with congenital heart disease at age 11 months, and the echocardiogram showed arterial ductus arteriosus, with a left-to-right shunt at the level of the arteries. Precedent III.10 was a twin of Proband III.9 who was diagnosed with congenital heart disease at age 11 months, in whom the echocardiogram revealed an arterial ductus arteriosus, an unenclosed patent ductus arteriosus, and a left to right shunt at the level of the arteries (second figure). III.8 was diagnosed with congenital heart disease at age 15, but echocardiography in this study showed no abnormalities. No cardiac abnormalities were detected in any of his parents, grandparents, or maternal grandparents. We performed whole-exome sequencing on CHD sufferers and their unexpressing family members to investigate the genetic causes of CHD in this family line. Exome sequencing identified 4 mutation sites in this family line. The variant c.3245A>G (p.His1082Arg) of the AMER1 gene was consistent with concomitant X-chromosome recessive inheritance, the variant c.238G>C (p.Val80Leu) of the KCNE1 gene was consistent with autosomal accessory inheritance, and the other 2 variants did not conform to the law of the mode of inheritance of the disease. Outcomes: The first identified variant, c.3245A>G (p.His1082Arg) of the AMER1 gene, with X-chromosome recessive inheritance, and the variant c.238G>C (p.Val80Leu) of the KCNE1 gene, which has been reported as autosomal dominant, may be the causative agent of CHD in this family line. These findings broaden the genetic scope of congenital heart disease and could help in the development of targeted drugs for the treatment of congenital heart disease.


Introduction
Congenital heart disease (CHD) is a malformation resulting from abnormal development of the heart and great vessels during embryonic life. [1]CHD includes malformations of the heart wall, valves, and blood vessels.The prevalence of CHD as the most common human birth abnormality is 0.8% to 1.2%. [2]Epidemiologic surveys have shown that the incidence

This research was funded by the National Natural Science Foundation of China (grant numbers 31960149), Zhiyuan Talent Program of Inner Mongolia Medical University (grant number ZY0120024), undergraduate science and technology innovation project of Inner Mongolia Medical University (grant number YCPY2021082) and doctoral research start-up fund project of Hohhot First Hospital (grant number 2023SYY(BS)001).
Informed consent was obtained from all individual participants included in the study.Informed consent was obtained from the parents of the 3 children in the family line.The authors affirm that human research participants provided informed consent for publication of the images in Figures 1, 2, and 3.

The authors have no conflicts of interest to disclose.
The datasets generated during and/or analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request.
This study was performed in line with the principles of the Declaration of Helsinki.Approval was obtained from the Ethics Committee of the First Hospital of Hohhot.
of CHD is increasing annually in China. [3]Despite advances in medical care, CHD remains the leading cause of death in infants and children in both developed and developing countries. [4]The causes of CHD are intricate and complex.Although scientists have conducted in-depth research on the pathogenesis of CHD, only 20% of the genes responsible for CHD have been identified, and most CHD is still unexplained, which has greatly limited the development of clinical treatments for CHD.
[7][8] Thanks to findings from animal experiments and medical genetics, about 400 genes have been linked to the development of CHD.Disorders associated with the AMER1 gene and the KCNE1 gene can partially exhibit a CHD phenotype.The AMER1 gene is located at human chromosome Xq11.2,with a total length of 3408 bp, encoding APC membrane recruitment protein 1 (AMER1), with a total length of 1135 AA.According to the Online Mendelian Inheritance in Man database, mutations in AMER1 cause X-linked dominant osteopathia striata with cranial sclerosis (OS-CS). [9]OSCS is a rare X-linked skeletal dysplasia typically associated with longitudinal striae in the metaphyseal region of the long bones and thickening of the skull, as well as macrosomia, dysmorphic facial features, and cardiac septal defects.The relevance of the AMER1 gene to CHD is that it acts mainly through negative regulation of the Wnt signaling pathway. [10]The KCNE1 gene is located at human chromosome 21q22.1-21q22.2,and the encoded product is the 13th subunit of the potassium ion channel (Mink protein), a single-transmembrane protein.Mutations in the KCNE1 gene are the most common cause of congenital defects in long QT syndrome, [11] a heart condition.Variant c.238G > C (p.Val80Leu) of the KCNE1 gene has been associated with long QT syndrome. [12,13]KCNE1 regulates the potassium voltage-gated channel subfamily Q member 1 (KCNQ1) channel in a variety of ways [14] : by slowing voltage-stimulated channel activation, increasing conductance, and eliminating channel inactivation. [15]KCNQ1, a voltage-gated potassium channel composed of 676 amino acids, is essential for cardiac action potential repolarization.
In 1 Chinese family with frequent CHD, we performed whole-exome sequencing of preexisting and unaffected family members.Our analyses using bioinformatics, online software prediction, and genetic pattern analysis, we concluded that variant c.3245A>G (p.His1082Arg) of the AMER1 gene, which is associated with X-chromosome recessive inheritance, and c.238G>C (p.Val80Leu) of the KCNE1 gene, which is inherited in an autosomal accessory manner, may cause the CHD in this family line.And the AMER1 c.3245A>G was discovered for the first time.This enlarges the genetic spectrum of congenital heart disease and provides new ideas for its diagnosis and treatment.

Case presentation
A three-generation CHD Han Chinese family line was recruited from Ordos Central Hospital.All family members were clinically evaluated by reviewing their medical history, performing a physical examination, and reviewing their medical records.This family line consisted of 3 generations totaling 10 individuals (Fig. 1).All participants underwent a detailed physical examination, including auscultation of the precordial region, checking for the presence of other clinical signs and symptoms associated with CHD, and cardiac ultrasound to confirm or exclude the diagnosis of CHD.Proband III.9 was diagnosed with congenital heart disease at age 11 months, and the echocardiogram showed arterial ductus arteriosus, with a left-to-right shunt at the level of the arteries.Precedent III.10 was a twin of Proband III.9 who was diagnosed with congenital heart disease at age 11 months, in whom the echocardiogram revealed an arterial ductus arteriosus, an unenclosed patent ductus arteriosus, and a left to right shunt at the level of the arteries (Fig. 2).III.8 was diagnosed with congenital heart disease at age 15, but echocardiography in this study showed no abnormalities.No cardiac abnormalities were detected in any of his parents, grandparents, or maternal grandparents.Peripheral blood samples were collected from patients and their family members.The study protocol was approved by the Medical Ethics Committee of the First Hospital of Hohhot.Written informed consent was obtained from all participants.

Whole-exome sequencing and bioinformatics analysis
Blood sample collection and DNA extraction: Subjects were fasted overnight, 2 mL of peripheral venous blood was drawn, blood specimens were treated with EDTA for anticoagulation, blood was centrifuged at 3000 r/min for 10 minutes, and then the genomic DNA of the blood samples was extracted with the Bomade Whole Blood Genome Extraction Kit.The concentration and purity of the DNA were determined using a DNA concentration detector (NANODROP2000, Thermo), and the DNA was preserved at −80 °C.
Whole-exome sequencing: Whole-exome sequencing of the congenital family line was completed at the Huada Medicine Laboratory in Shenzhen, China.The genomic DNA of the subject's blood was first broken up, and libraries were prepared.Then, the DNA of the target exons and adjacent shear regions were captured and enriched by the Roche KAPA HyperExome chip.Finally, the MGISEQ-2000 sequencing platform was used for variant detection.The quality control index of the sequencing data was as follows: the average sequencing depth of the target region was ≥180×, among which the proportion of loci with an average depth > 20× in the target region was >95%.Sequenced fragments were aligned with the UCSC hg19 human reference genome (http://genome.ucsc.edu/)by the Burrows-Wheeler Aligner tool (BWA, version 0.7.15) to remove duplicates.Base mass value correction for SNV, INDEL and genotype detection was performed using the Genome Analysis Toolkit (GATK, version 3.3.0).ExomeDepth was used for copy number variation detection at the exon level.Variants with a minimum gene frequency greater than or equal to 1% in databases such as Thousand Genomes (Phase3), ESP6500 (V2), ExAC (r0.3.1),GnomAD (r2.0.1),GnomAD-EAS, etc., were excluded.
Bioinformatics analysis: Prediction of nonsynonymous SNPs was performed by SIFT, MutationTaster, and Condel, and the effect of splice site variants on splicing was predicted by SpliceAI (1.3), dbscSNV_ ADA, and dbscSNV_ RF.Nucleic acid conservation was predicted by PhyloP Vertebrates, PhyloP Placetal Mammals, and GERP++.Variant pathogenicity was classified according to the American College of Medical Genetics and Genomics, and American Molecular Pathology Society guidelines for interpretation of sequence variants, with reference to the ClinGen Working Group on Interpretation of Sequence Variants and the British Academy of Clinical Genome Sciences for a refined interpretation of the guidelines.
The probands, as well as their mother and grandmother, carried a variant in the AMER1 gene, which was not found in any other family member (Fig. 3A).The analysis of the gene for this variant based on its position on the chromosome indicated that the inheritance of this variant conformed to the X-chromosome recessive mode of inheritance, the probands all being hemizygous, the mother and grandmother heterozygous, and the rest of the family members wild-type, which is in accordance with the law of the mode of inheritance of the disease.A variant in the KCNE1 gene was found in the mother of the probands, but she did not have the disease (Fig. 3C), while all 3 children carrying the same genotype had the disease, suggesting that the variant   was a de novo mutation occurring in the mother and that the mode of inheritance was consistent with autosomal sex dependent inheritance, as analyzed by the mother's and the three children's genetic properties.The AMER1 c.3245A>G variant at codon 3245 leads to substitution of arginine for histidine, and GERP++ is predicted to be conserved, suggesting that this variant may be clinically significant.This variant was not found in several databases: the Thousand Genomes Project, ESP6500, ExAC, GnomAD, and GnomAD-EAS.There are no reports of pathogenicity associated with this variant.According to the Sequence Variant Interpretation Guidelines, c.3245A>G is categorized as a variant of undetermined significance (PM2 + BP1).The KCNE1 c.238G>C variant is a substitution of valine by isoleucine at codon 238, which is predicted to be conserved by GERP++, suggesting that a mutation at this site may be clinically significant.This variant has low frequency in the ESP, Thousand Genomes, EXAC, GnomAD, and GnomAD-EAS databases, and its pathogenicity has been reported.According to the Sequence Variant Interpretation Guidelines, c.238G>C is categorized as a variant of undetermined significance (PM1 + PM2 + PP2).Disorders associated with the AMER1 gene and the KCNE1 gene can have a cardiac dysplasia phenotype, so variants in the AMER1 gene and variants in the KCNE1 gene in this family line may be responsible for their CHD.

Discussion and conclusion
In the Chinese family with preexisting heart disease that we studied here, the first proband presented with arterial ductus arteriosus and left-to-right shunting at the arterial level.By whole-exome sequencing, we identified variants in 4 genes in the family line.Those that were analyzed to fit the pattern of disease inheritance were a new hemizygous missense variant, c.3245A>G (p.His1082Arg), found in the AMER1 gene and a new heterozygous mutant, c.238G>C (p.Val80Leu), found in the KCNE1 gene.
AMER1 is a potential dose-sensitive candidate gene for congenital cardiac anomalies. [16]The AMER1 protein contains 3 APC-binding domains (A1-A3), 2 membrane-localized structural domains (M1 and M2) that can absorb APCs from microtubules to the plasma membrane, and a structural domain consisting of a repetitive arginine-glutamic acid-alanine motif that directly interacts with the armadillo repeats of β-conjugated proteins and is a negative regulator of Wnt signaling during development(Fig.4A). [17]Striated osteomalacia with craniosclerosis caused by variants in the AMER1 gene can present with a congenital cardiac dysplasia phenotype in some patients. [18]ther authors have found an increase in the number of copies of the AMER1 gene in a male patient with mental retardation, congenital heart malformation, and obesity. [16]AMER1 Figure 3. Four genes identified by whole-exome sequencing that may be associated with CHD and the genotypes of the variants carried by each family member, as well as the corresponding amino acid changes.
can block typical Wnt signaling by inducing proteasomal degradation of β-conjugated proteins, and studies in zebrafish and Xenopus laevis have also demonstrated an inhibitory role for AMER1 in Wnt signaling. [19]Disruption of Wnt signaling is associated with congenital heart defects in animal models. [20]hese findings emphasize the importance of the AMER1 protein for cardiovascular development.Thus, it is possible that variants in AMER1 contribute to CHD in humans.The AMER1 gene variant site identified in this study is not within a known important structural domain, and its effect on protein function needs to be further investigated.The product encoded by the KCNE1 gene is the 13th subunit of the potassium channel (Mink protein), a single-transmembrane protein that regulates the voltage-gated potassium channel KCNQ1 by slowing activation and enhancing channel conductance.The KCNQ channel contains a voltage-sensitive structural domain (VSD) and a pore structural domain.Upon activation, the positively charged voltage sensor of the VSD senses changes in membrane potential and moves outward within the membrane, opening the pore through VSD-pore structural domain coupling.The transmembrane structural domain of KCNE1 slows channel activation by connecting the voltage sensor to the S4-S5 connector of the pore structural domain and restricting its movement(Fig.4B). [15]otassium ion channels are distributed in large numbers across the myocardial cell membrane, and the changes in their currents are closely related to myocardial cell membrane excitability.Many patients with malignant arrhythmias, such as nonfamilial arrhythmias and Jervell syndrome, have mutations in the KCNE1 gene. [21]Therefore, variations in the KCNE1 gene may cause arrhythmias by affecting potassium ion channels, which can lead to heart disease.Further studies are needed to identify the unknown structural domains of the KCNE1 protein.
In conclusion, the novel hemizygous missense variant c.3245A>G (p.His1082Arg) in the AMER1 gene and the de novo mutation c.238G>C (p.Val80Leu) in the KCNE1 gene that we identified in this lineage may be the causative variants of their CHD.It is unclear how either variation contributes to the prevalent heart disease phenotype.Therefore, functional experiments in vitro and in vivo are needed to elucidate the exact mechanism linking these missense variants to their heart disease, and studies with larger sample sizes are needed to identify more cases of prevalent heart disease associated with AMER1 and KCNE1 variants.
Several other genetic variant loci that we identified in this whole-exome sequencing result were analyzed in this family line and did not fit the pattern of their CHD inheritance pattern.However, variants of the PUF60 and MED13L genes cause disorders that can include phenotypes associated with CHD: PUF60 gene-associated disorders such as Verheij syndrome and prenatal anomalies, and MED13L gene-associated disorders such as anomalous type 1 of the right great artery, mental retardation and typical facial features with or without heart defects, among others.It is possible that interactions between these genes or other factors play a role in these patients, so these mutation loci could be further validated in other family lines or by expanding the sample size to validate them in the population.The findings of such studies would further expand the genetic spectrum of prevalent heart disease.

Figure 1 .
Figure 1.Genealogical map of the family lineage of congenital heart disease.Boxes are males, circles are females, and black represents patients with congenital heart disease.

Figure 2 .
Figure 2. Ultrasonographic findings of the twin probands in this family line.(A and B) Proband III.9, ultrasonographic images showed an unobstructed arterial duct with a left-to-right shunt at the level of the arteries, which was diagnosed as CHD.(C and D) Proband III.10, ultrasonographic images showed an unobstructed arterial duct with a left-to-right shunt at the level of the arteries, which was diagnosed as CHD.
Ectopic right circumflex artery type 1/AD Intellectual impairment and typical facial features with or without heart defects/AD AA = amino acid, AD = autosomal dominant inheritance, AR = autosomal recessive inheritance, B = brother, F = father, M = mother, P = proband, XL = X-linked inheritance.

Figure 4 .
Figure 4. Schematic representation of the structural domains of AMER1 and KCNE1 proteins and the locations of the mutation sites in the structural domains of the proteins identified by whole-exome sequencing in this lineage.(A) AMER1 contains 3 APC-binding domains, A1-A3, that have no obvious sequence similarity to each other, 2 N-terminal phosphatidylinositol (4,5) bisphosphate-binding domains (M1 and M2), which can take up APC from the microtubule to the plasma membrane and are membrane-localized structural domains, and an arginine-glutamate-alanine (REA) motif that interacts directly with armadillo repeats of β-connexin and negatively regulates Wnt signaling.(B) KCNE1 is a single-transmembrane protein that regulates the voltage-gated potassium channel KCNQ1 by slowing its activation and enhancing its conductance.Upon activation, the positively charged voltage sensor of the VSD senses changes in membrane potential and moves outward within the membrane, opening the pore through VSD-PD coupling.The transmembrane structural domain (TM) of KCNE1 slows the activation of the channel by connecting the voltage sensor to the S4-S5 junction of the pore structural domain and restricting its movement.